Antioxidant and Antimicrobial Activity of 5-methyl-2-(5-methyl-1,3-diphenyl-1H-pyrazole-4-carbonyl)-2,4-dihydro-pyrazol-3-one.

Cycloaddition of nitrile imines 4 generated in situ by the catalytic dehydrogenation of diphenyl hydrazones 3 using Chloramine-T (CAT) as oxidant in glacial acetic acid with enolic form of ethyl acetoacetate 5 afforded Ethyl 3-aryl-5-methyl-1-phenyl-1H-pyrazol-4-carboxylate 6 in 80% yield. The said pyrazoles 6 refluxed with 80% hydrazine hydrate using absolute alcohol as solvent for about 2-3 hours to produce the respective 5-methyl-1,3-diphenyl-1H-pyrazole-4-carboxylic acid hydrazide 7. The alcoholic solution of pyrazole acid hydrazides on heating with ethyl acetoacetate 5 to give the 5-methyl-2-(5-methyl-1,3-diphenyl-1H-pyrazole-4-carbonyl)-2,4-dihydro-pyrazol-3-one 8. The synthesized compounds were found to exhibit good antimicrobial and antioxidant activity as evaluated by 1,1-diphenyl-2-picryl Hydrazyl (DPPH) radical scavenging, reducing power and DNA protection assays.


Bioactivity studies of synthesized pyrazole derivatives
Antioxidant activity Scanning electron microscopic studies of erythrocyte oxidation. Erythrocytes were obtained from healthy donors. Heparinized blood was centrifuged at 1000 g for 15  min. After removal of plasma and buffy coat, the erythrocytes were washed thrice with PBS (20 mM, pH 7.4, NaCl -0.9%) at room temperature and resuspended in PBS four times its volume for subsequent analysis (24). Erythrocytes were preincubated with samples (0.5 mg/ mL) 5-methyl-2-(5-methyl-1,3-diphenyl-1H-pyrazole-4-carbonyl)-2,4-dihydropyrazol-3-one 8a-g which were dissolved in PBS containing 0.25% DMSO for 5 min. These concentrations of DMSO were found to have no effect on erythrocytes. Then hydrogen peroxide (30 mM), ferric chloride (80 µM) and ascorbic acid (50 µM) were added and incubated at 37°C for 1 hour. The reaction mixture was gently shaken while being incubated (25). Then the cells were fixed overnight at 4°C with glutaraldehyde in normal saline, reaching a final fixation concentration of about 2.4%. The cells were washed in saline solution and then dehydrated using ascending grades of alcohol (10-100%). Few drops of each sample were placed on A-1 glass cover slips, air dried at room temperature, gold coated and examined in a scanning electron microscope.
DPPH radical scavenging assay. The effect of the samples 8a-g in addition to the standard antioxidant butylated hydroxyl toluene (BHT) on DPPH radical was estimated according to the method of Lai et. al (26). Samples solubilized in methanol (0-50 µg/mL for samples 8a-g; 0-5 µg/mL for BHT) in 200 µL aliquot was mixed with 100 mM Tris-HCl buffer (800 µL, pH 7.4) and then added 1 mL of 500 µM DPPH in ethanol (final concentration of 250 µM). The mixture was shaken vigorously and left to stand for 20 min at room temperature in the dark. The absorbance of the resulting solution was measured spectrophotometrically at 517 nm. The capability to scavenge DPPH radical was calculated using the following Equation 1.
Measurement of reducing power. The reducing power of samples 8a-g was determined according to the method (27) of Yen and Chen. The samples 8a-g (0-50 µg/mL) were mixed with an equal volume of 0.2 M phosphate buffer, pH 6.6 and 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min. Then an equal volume of 10% trichloroacetic acid was added to the mixture and then centrifuged at 5000 g for 10 min. The upper layer of solution was mixed with distilled water and 0.1% ferric chloride at a ratio of 1:1:2 and the absorbance were measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power. DNA protection assay. DNA protection ability of samples 8a-g was performed using lambda phage DNA (28). Briefly, λ phage DNA (0.6 µg) was subjected to oxidation using Fenton's reagent (0.3 mM hydrogen peroxide, 0.5 µM ascorbic acid and 0.8 µM ferric chloride) in presence and absence of the sample (0.2 mg) for 2 hours at 37°C. The samples 8a-g was subjected for electrophoresis (Submarine electrophoresis system, Bangalore Geni, Bangalore, India) on 1% agarose for 2 hours at 50 volts DC. Gels were stained with ethidium bromide (0.5 µg/mL) and documented (Herolab, Germany). statistical analysis. All the experiments were carried out in triplicates (n=3) and the results are expressed as mean ± standard deviation (SD).
Antimicrobial activity Antibacterial activity assay by paper disc diffusion method (29). Synthesized Pyrazoles (8a-g) were screened (dose of 100µg) for their antibacterial activity against Gram-negative bacteria Escherichia coli (E. coli) and Gram-positive bacteria (S. aureus) using filter paper disc method. Plates inoculated with E. coli were incubated for 48 hr and plates inoculated with S. aureus for 24 hr respectively at room temperature. Streptomycin sulphate was used as a standard. After the period of incubation the inhibition zones were measured in mm and results obtained are shown in Table 1.
Antifungal activity assay. All the compounds were also screened (dose of 100µg) for their antifungal activity against C. albicans and A. niger using Griseofulvin as a standard. The results are shown in Table 1
In typical reaction, a mixture of aldehyde hydrazone 3a (2.35 g, 12.0 mmoles), excess of ethyl acetoacetate 5 (2.6 g, 20.0 mmoles) and CAT (3.94 g, 14.0 mmoles) in glacial acetic acid and stirred at room temperature for about 2-3 hours. After the usual work up, the reaction afforded 6a as light yellow oil in 80% (2.93 g) yield. IR, 1 H NMR, 13 C NMR, MS studies and elemental analysis provide the structural proof for the products. For instance, in IR spectra, the peak expected due to -OH group in the region 3550-3640 cm -1 was found absent and it shows ester carbonyl stretching frequency at 1716-1728 cm -1 and a C=N frequency at 1608-1632 cm -1 . In 1 H NMR spectra, the signal due to -OC 2 H 5 protons appears as a quartet in the region δ 4.12-4.31 ppm, (2H for -O-CH 2 -.group) and a triplet in the region 1.18-1.30 ppm, (3H for -OCH 2 -CH 3 ), while the vinylic -CH 3 protons appear as a singlet in the region δ 2.68-2.75 ppm. is probably due to deshielding by -CO-OC 2 H 5 group. These observations support the formation of the cycloadducts 6 with the loss of water molecule. In 13 C NMR spectra, the -C 3 and -C 4 -carbon appear as singlet (decoupled) in the region δ 160.82-161.14 and δ 108.32-118.86 ppm respectively, while C 5 -carbon appear as singlet in the region δ 176.14-176.26 ppm. All cycloadducts showed MH + as a base peak in the mass spectra and significantly stable molecular ion peaks with the relative abundance ranging from 20-90%, which strongly favors the formation of the cycloadducts.

Effect of samples 8a-g on Erythrocyte Oxidation
The effect of samples 8a-g was studied for their preventive effect on erythrocyte oxidation using the method as described in materials and methods. The scanning electron micrographs (Figure 4) show the protective ability of 8a and 8f samples on erythrocyte membrane oxidation. As compared to normal erythrocytes, erythrocytes treated with hydrogen peroxide showed the appearance of echinocytes and also agglutination indicating damage to the cell membrane. In 8a-g samples the presence of normal cells can also be seen in addition to oxidized cells indicating the protective role of these compounds. The protection may not be comparable to that of the normal cells, but compared to the oxidized erythrocytes protection by the tested compound is evident.

Antioxidant activity of samples 8a-g
The antioxidant activity of samples 8a-g was evaluated by DPPH radical scavenging, reducing power and DNA protection assays. The free radical scavenging ability of samples 8a-g was evaluated by DPPH scavenging model system using the Equation 1 ( Table 2). All the samples showed free radical scavenging ability, but when compared with the standard antioxidant the samples tested showed 50% lesser activity. These results indicate the potential electron donating ability of samples.
In addition, reducing power of samples 8a-g was also evaluated ( Table 3) for their ability to reduce ferric chloride and potassium ferricyanide complex. At the initial concentrations (10-20 µg/mL) there was no significant differences in the activity were observed. However, as the concentration was increased (30-50 µg/mL) 8f showed higher reducing power and 8a showed lower reducing power. The increased absorbance at 700 nm indicated the presence of reducing power.
Also, DNA protective ability of 8a-g were evaluated on lambda phage DNA oxidation ( Figure 5). The hydroxyl Normal Oxidized 8f treated + Oxidised 8a treated + Oxidised   radical generated by Fenton's reagent caused DNA fragmentation with increase in its electrophoretic mobility and thereby the fragments have run out of the gel (lane 2). Upon treatment of 8f and 8a prior to oxidation the extent of DNA damage was minimized. As evidenced by gel documentation analysis, compared to native DNA (lane 1), higher protection (45-50%) was observed in 8f treated sample (lane 3), while 40-45% protection was observed for 8a (lane 4).

Observation
It was observed that the pyrazoles (8a-g) derivatives showed less antibacterial and antifungal activity. The antibacterial activity of 8f, 8b, 8e, 8g, 8c, 8d and 8a shows Gram-negative bacteria Escherichia coli (E. coli) and Gram-positive bacteria (S. aureus). The antibacterial and antifungal activity of 8f is more compared to other. This indicates that, as the size and substituents increases the antibacterial and antifungal activity increases.

EXPERIMENTAL typical procedure for the preparation of Ethyl 5methyl-1,3-diphenyl-1H-pyrazole-4-carboxylate (6a)
A mixture of benzaldehyde hydrazone (3 a, 2.35 g, 12.0 mmoles), excess of freshly distilled ethyl acetoacetate (5, 2.6 g, 20.0 mmoles) and CAT (3.94 g, 14.0 mmoles) in glacial acetic acid (25 ml) were stirred at room temperature for 2-3 hours. The progress of the reaction was monitored by TLC. After the completion of the reaction the residual mass was extracted into ether (25 ml), washed successively with water (2 × 20 ml), 1N NaOH (1 × 10 ml), brine solution (2 × 15 ml) and dried over anhydrous sodium sulphate. Evaporation of the solvent afforded crude oily substance, which, in TLC (chloroform : acetone : 7 :1 v/v) gave one major spot with R f value 0.66, two minor spots with R f values 0.58 and 0.52 corresponding to the product and starting materials respectively. Purification was done by column chromatography using benzene : ethyl acetate